23

L-type Amino Acid Transporter-1 Specific Imaging Agent
茨城県立医療大学紀要ASVPI

第 24 巻

Original Article

Volume 24

An L-type Amino Acid Transporter-1 Specific Imaging Agent:
Structure-function Relationships of Radioiodinated Tyrosine
Derivatives
Naoto Shikano1), Yoshikatsu Kanai2), Keiichi Kawai3), Jun Inatomi2),
Do Kyung Kim2), Ayako Ikeda1), Hitoshi Endou2)
1)

Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan3)
Division of Health Science, Graduate School of Health Sciences, Kanazawa University2)

Abstract
Introduction: We recently reported the high isoform selectivity of 3-125I-α-methyl-L-tyrosine (125I-IMT) to L-type amino acidtransporter-1 (hLAT1) of the two system L1 membrane transporters. Additional tyrosine derivatives were examined herein toidentify a more hLAT1-selective compound.
Methods: An uptake study using 4-125I-L-meta-tyrosine (4-125I-mTyr), 6-125I-L-meta-tyrosine (6-125I-mTyr) and 3-125I-L-tyrosine(3-125I-Tyr) was performed using transporter-expressing Xenopus laevis oocytes. Oocytes were injected with 17.6 ng of hLAT1or hLAT2 complementary RNA (cRNA) and 7.4 ng of human 4F2hc cRNA in a molar ratio of 1:1. Two days after injection,
uptake of the tyrosine derivatives was measured in Na+-free uptake solution containing 18.5 kBq of noncarrier-added tyrosine
derivative by determining the radioactivity in the oocytes after incubation for 30 min at room temperature.
Results: The order of higher selectivity was 4-125I-mTyr > 125I-IMT > 3-125I-Tyr > L-14C(U)-Tyr ≑ 6-125I-mTyr.

Conclusion: hLAT exhibited the highest transport specificity towards 4-125I-mTyr of the five tyrosine derivatives tested.
Key words: system L1, tyrosine derivatives, human L-type amino acid transporter 1, human 4F2hc, Xenopus laevis
oocyte1. Introduction

respectively, based on the standards established by the
Human Gene Nomenclature Committee.2–5) The SLC7

Several amino acid transport systems have beenfamily is further divided into the cationic amino aciddescribed, of which one, system L, is a Na -independenttransporter family and the L-type amino acid transportertransport system and a major route for providing cells(LAT) family.2–5) The two known isoforms of system L1with large neutral amino acids, including branched andbelong to the LAT family and are named type 1 (LAT1)

aromatic amino acids.1) System L1 and L2 (components ofand type 2 (LAT2). The LAT1 and LAT2 transporters ofsystem L) genes are classified into two families of solutesystem L1, but not the LAT3 and LAT4 transporters ofcarriers (SLCs), the SLC7 and SLC43 families,

system L2, form a heterodimeric complex with an+Address: Naoto Shikano. Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi,
Inashiki-gun, Ibaraki 300-0394, Japan
Phone: +81-29-840-2217, Fax: +81-29-840-2317, E-mail address: sikano@ipu.ac.jp

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茨城県立医療大学紀要24

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2. Materials and Methods

additional protein, the heavy chain of the 4F2 antigen(CD98; 4F2hc/SLC3A2), which is required for LAT1/2
transport function. LAT1/2 and LAT 3/4 also differ in that

2.1. Labeled Compounds

LAT1 and LAT2 selectively transport neutral amino acids

Reagent grade chemicals (Aldrich Chemical Co.,by obligatory exchange mechanisms, whereas LAT3 and

Milwaukee, WI) were used throughout. 125I-NaI (8.1 ×LAT 4 do not display obligatory exchange mechanisms.

1019 Bq/mol) was obtained from Amersham Pharmacia

All these transporters show partly overlapping expression

Biotech (Buckinghamshire, U.K.). Noncarrier-added

patterns and substrates, although some differences in their

4-125I-mTyr, 6-125I-mTyr and 3-125I-Tyr were prepared byexpression and substrates have been reported.2–5)

theWe are currently studying radioiodinated tyrosine
derivativeswith

theintentof

selectivityof

chloramine-Tmethod

describedpreviously.

6,7)

developingradiopharmaceuticals.6-10) Recently we reported thetransporter

conventional

2.2. Uptake Studies with X. laevis Oocytes

3-123/125I-iodo-α-methyl-l-Animal experiments were approved by the ethics

tyrosine (123/125I-IMT), which is derived from tyrosine.

committees of affiliated universities. In vitro transcription

I-IMT is transported by two isoforms of system L1

was performed as described previously11-13) to obtainhuman L-type amino acid transporters (LATs), designatedcomplementary RNAs (cRNAs) for hLAT1 and hLAT2

hLAT1 and hLAT2. Human LAT1 and hLAT2 must bindusing T3 RNA polymerase in pBluescript II SK−

with the heavy chain of human 4F2 cell-surface antigen(Stratagene, La Jolla, CA) linearized with Xho I, and T712,13)(h4F2hc) for system L1 to be functionally expressed.

RNA polymerase for h4F2hc in plasmid pZL1 (Invitrogen,In this pilot test, we performed uptake studies using

Carlsbad, CA) linearized with BamHI. Expression studies

123/12511)

4-125I-L-meta-tyrosine

6-125I-L-meta-

were conducted by injecting 17.6 ng of hLAT1 or hLAT2tyrosine (6-125I-mTyr) and 3-125I-L-tyrosine (3-125I-Tyr)cRNA and 7.4 ng of h4F2hc cRNA (molar ratio, 1:1) into(Fig. 1), hLAT1 or hLAT2, and h4F2hc-coexpressing

X. laevis oocytes. The control group consisted of X. laevis

Xenopus laevis oocytes, and compared the results obtained

oocytes injected with water instead of cRNA solution.

with those previously reported for

Uptake of radiolabeled amino acids was measured 2 daysTyr.

(4-125I-mTyr),

123

I-IMT and l- C(U)14

after injection by placing the oocytes in a Na+-free uptake11)

solution (100 mmol/L choline chloride, 2 mmol/L KCl, 1Ammol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L Hepes, 5B

HO

H

1414

C(U)-L-tyrosine ( C-Tyr)

CHOH

I
125

I-L-tyrosine (3-I-Tyr)

H

COOH
NH2HO
125

ICH3I-mTyr)I-α-methyl-L-tyrosine (Labeling efficiency was >80%; after purification, theradiochemical purities of theI-labeled tyrosine

125

COOH

derivatives were >95% and their specific radioactivity

NH2was >8.1 × 1019 Bq/mol. Figure 2 shows the results

125

143. Results

COOH

125

I-L-meta -tyrosine (6-I3-tyrosine derivative.

I-rabeled

125

NH2HO125

125

NH2H

COOHE HOtemperature) containing a 18.5 kBq/mLI-mTyr)D

6-COOH

125

I-L-meta -tyrosine (4-4-NH2125

3-mmol/L Tris, pH 7.4; incubation for 30 min at room

II-IMT)obtained in this study for 4-125I-mTyr, 3-125I-Tyr and125

Fig. 1 Chemical structurers of l- C(U)-Tyr (A), 4- I-L-metatyrosine (4-125I-mTyr) (B), 3-125I-L-tyrosine (3-125I-Tyr) (C),6- 125I-L-meta-tyrosine (6- 125I-mTyr) (D) and 3- 125I-iodo-αmethyl-l-tyrosine (125I-IMT) (E).

6-125I-mTyr, as well as the results obtained previously with
I-IMT and l-14C(U)-Tyr.11) The uptakes of 125I-IMT,

125

4-125I-mTyr and 3-125I-Tyr by X. laevis oocytes via thePresented by Medical*Online

25

L-type Amino Acid Transporter-1 Specific Imaging Agent

4. Discussion

hLAT1-h4F2hc heterodimer were higher than their uptake
by the hLAT2-h4F2hc heterodimer (P < 0.005), whereas
the uptakes of 6-125I-mTyr and l-14C(U)-Tyr were not

Rat LAT1 was the first LAT1 to be identified, by Kanai

significantly different between hLAT1-h4F2hc- andet al. in 1998.14) The relationship between LAT1 and cellhLAT2-h4F2hc-coexpressing X. laevis oocytes (Fig. 2).

proliferation, tumor malignancy, and the transport ofThe transport activity via hLAT1 followed the order (fromessential amino acids has been described.4) Manyhigh to low): 3-125I-Tyr > 4-125I-mTyr > L-14C(U)-Tyr >malignant tumors highly express LAT1, such as tumors

I-IMT > 6- I-mTyr.

125

resulting from colon, breast, head and neck, genital,

125

gastric, thyroid and thymic cancers, and soft-tissue
sarcoma and hepatocellular carcinoma.15) LAT1 is highly

2,000

**

20,00015,00010,000

**

5,000

5,000

L-boronophenylalanine,

600

*

for

theandnatural

500

specific inhibitor of the LAT1 transport of activatedleukocytes),16) and for the diagnostics 123I-IMT and L-3[18F] fluoro-alpha-methyl tyrosine20).

**

400

LAT1 and LAT2 are members of the system L1 aminoacid transporters and selectively transport neutral amino

300

acids by obligatory exchange mechanisms.4,12) These200100

transporters exhibit broad substrate selectivity for various
hLAT2

hLAT1

hLAT2

E 5,000
cpm/oocyte/30 minMelphalan18)

immunosuppressant brasilicardin A (a very high affinity

04,000

KYT-0353,17)
19)

00
hLAT1

cpm/oocyte/30 min

25,000including

DC 30,000

LAT1 is an important target for various therapy drugs,

10,000hLAT2015,000hLAT24,000cells.16)
**

hLAT1

**

cpm/oocyte/30 min*

cpm/oocyte/30 min

6,000

expressed on the membranes of activated white blood

B 20,000hLAT1

cpm/oocyte/30 min

A 8,000

neutral amino acids but LAT1 and LAT2 have differentroles.8) For example, LAT1 exhibits higher affinity (Km =20–40 μmol/L) but lower capacity toward Leu, Ile, Phe,Met, Tyr, His, Trp, and Val, and accepts the d-isomers of**

3,000

Leu, Phe, and Met as substrates.12) In contrast, the2,000

heterodimeric complex of LAT2 and 4F2hc is involved in

1,000

the transcellular transport of neutral amino acids throughthe epithelia and blood-tissue barriers.13) Furthermore,hLAT2

hLAT10LAT1 prefers larger neutral amino acids with branched orFig. 2 Comparison of isoform selectivity of hLAT1 or hLAT2
and h4F2hc-coexpressing X. laevis oocytes for l- 14C(U)Tyr (A), 4-125I-mTyr (B), 3-125I-Tyr (C), 6-125I-mTyr (D) and125
I-IMT (E). The controls were water-injected oocytes. Uptakeof each radiolabeled amino acid was measured in Na+-freeuptake solution containing 18.5 kBq radiolabeled amino acid.
4-125I-mTyr transport showed hLAT1-h4F2hc selectivity. Datarepresent the average +/- s.e.m. of the uptake of each labeledamino acid. A significant difference from the control value isindicated by *P<0.05; **P<0.005. The data for 125I-IMT andl-14C(U)-Tyr are reprinted with permission from Ref. 11.

aromatic side chains, whereas LAT2 exhibits lower
affinity (Km = 30–300 μmol/L) but a higher capacity andremarkably broad substrate selectivity, including the small
neutral amino acids Gly, Ala, Ser and Thr.13)
We speculated that the increase in molecular weight orbulkiness caused by the introduction of an iodine atom
into the phenol ring of Tyr could change the affinity of Tyr
for these amino acid transporters (Fig. 1). The molecularweight of Tyr (or mTyr) and alpha-methyl tyrosine isabout 181 Da and 195 Da, respectively, and the atomic
weight of the stable isotope of iodine is about 127 Da.

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茨城県立医療大学紀要26

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Conflict of interest

Thus, the atomic weight of iodine accounts forapproximately 41% and 38% of the molecular weight ofC9H11NO3I (for 3-I-Tyr, 4-I-mTyr and 6-I-mTyr) andC10H13NO3I (for IMT), respectively. The present results

The authors declare that they have no conflict ofinterest.

indicate that the amino acid transporters exhibited
different specificities towards these molecularly altered
Acknowledgementamino acids (Fig. 2). Also, differences in the position of
the iodine and hydroxyl groups in the phenol ring, and thepresence of an alpha-methyl group, resulted in altered

The authors thank Tomoaki Murakami, Makoto Hira,

specificities of the system L1 amino acid transporters forMiya Kikuchi, Kohichi Hamazaki, Michiko Nishikido,

these derivatives.

Mayo Hakamada, and Rinko Sasaki (Ibaraki PrefecturalThe selectivity by hLAT1 followed the order (fromUniversity of Health Sciences) for their technicalhigh to low): 4-125I-mTyr > 125I-IMT > 3-125I-Tyr >assistance. This work was supported by Grants-in-Aid for

L-14C(U)-Tyr ≑ 6-125I-mTyr, suggesting the importance ofScientific Research (#26461801) from the Ministry ofstructure function relationships. The position of iodine in

Education, Science, Sports and Culture of Japan, and thethe phenol ring appears to be an important factor in theJapan Society for the Promotion of Science. Financial

system selectivity of radioiodinated tyrosine derivatives.

support was also provided by a Japan Atherosclerosis

In the present study, hLAT1 was highly selective for

Research Foundation Grant in 2018.

4- I-mTyr and 3- I-Tyr (Figs. 2 B and C), whereas
125

125

system L1 exhibited affinity for 6-125I-mTyr (Fig. 2 D).
References

LAT1 was highly specific for derivatives mono-iodinatedat the meta/para positions of the phenol ring, whereas
hLAT1 and hLAT2 exhibited reduced affinity for

1)

Christensen HN. Role of amino acid transport andderivatives mono-iodinated at the ortho position of thecounter-transport in nutrition and metabolism.

ring. System L1 exhibited affinity for alpha-methylated

Physiol Rev. 1990;70:43-77.

3- I-Tyr, as indicated by the results for alpha-methylated
1252)

3- I-Tyr (IMT) (Figs. 2 C and E).

Palacin M, Kanai Y. The ancillary proteins of HATs:

125

SLC3 family of amino acid transporters. Pflugers

Transport by hLAT1 exhibits the highest specificity forArch. 2004;447:490-494.

4-125I-mTyr and we are presently investigating 4-125I-mTyr3)

Kanai Y, Hediger MA. The glutamate/neutral amino

as a potential imaging agent because of its high tumor cellaciduptake via hLAT1, very high hLAT1 selectivity, and rapid

physiological and pharmacological aspects. Pflugers

clearance from blood.

Arch. 2004;447:469-479.

8)4)transporterfamily

SLC1:molecular,Verrey F, Closs EI, Wagner CA, Palacin M, Endou H,
Kanai Y. CATs and HATs: the SLC7 family of amino

5. Conclusionacid transporters. Pflugers Arch. 2004;447:532-542.
5)

Weissbach L, Handlogten ME, Christensen HN,

The heterodimeric complexes hLAT1-4F2hc andKilberg MS. Evidence of two Na-independenthLAT2-4F2hc are system L1 transporters. 4-125I-mTyrneutral amino acid transport systems in primary

was specifically transported by hLAT1-4F2hc, whereascultures

the parent L - C(U)-Tyr did not demonstrate isoform

1982;256:12006-12011.

14selectivity.

6)

ofrat

hepatocytes.

JBiolChem.

Kawai K, Flores LG 2nd, Nakagawa M, Shikano N,
Jinnouchi S, Tamura S, Kubodera A. Brain uptake ofiodinated L-meta-tyrosine, a metabolically stable

Presented by Medical*Online

27

L-type Amino Acid Transporter-1 Specific Imaging Agentamino acid derivative. Nucl Med Commun. 1999;
7)

20:153-157.

Chairoungdua A, Kim DK, Matsuo H, Kim JY,Flores LG 2nd, Kawai K, Nakagawa M, Shikano N,Miyamoto K, Takeda E, Endou H. Transport

Jinnouchi S, Tamura S, Watanabe K, Kubodera A. A

properties of a system y+L neutral and basic amino

newcerebralacid transporter. Insights into the mechanisms of

dopaminergic presynaptic function: 6-radioiodinatedsubstrate recognition. J Biol Chem. 2000;275:20787-

L-meta-tyrosine. J Cereb Blood Flow Metab. 2000;

20793.

radiopharmaceuticalforthe20:207-212.
8)9)14) Kanai Y, Fukasawa Y, Cha SH, Segawa H,15) Kaira K, Oriuchi N, Imai H, Shimizu K, Yanagitani

Shikano N, Kawai K, Flores LG 2nd, Nishii R,N, Sunaga N, Hisada T, Tanaka S, Ishizuka T, Kanai

Kubota N, Ishikawa N, Kubodera A. An artificial

Y, Endou H, Nakajima T, Mori M. l-type amino acidamino acid, 4-iodo-L-meta-tyrosine: biodistribution

transporter 1 and CD98 expression in primary andand excretion via kidney. J Nucl Med. 2003; 44:625-

metastatic sites of human neoplasms. Cancer Sci.

631.

2008; 99:2380-2386.

Shikano N, Kawai K, Nakajima S, Kubodera A,17) Oda K, Hosoda N, Endo H, Saito K, Tsujihara K,Kubota N, Ishikawa N, Saji H. Transcellular transport

Yamamura M, Sakata T, Anzai N, Wempe MF, Kanai

of 4-iodo-L-meta-tyrosine via system L across

Y, Endou H. L-type amino acid transporter 1

monolayers of kidney epithelial cell line LLC-PK1.

inhibitors inhibit tumor cell growth. Cancer Sci.

Nucl Med Biol. 2004; 31:477-482.

2010; 101:173-179.

10) Shikano N, Kotani T, Nakajima S, Ogura M,16) Usui T, Nagumo Y, Watanabe A, Kubota T, Komatsu

Nakazawa S, Sagara J, Kobayashi M, Baba T,K, Kobayashi J, Osada H. Brasilicardin A, a natural

Yamaguchi N, Kubota N, Kawai K. Radioiodinatedimmunosuppressant, targets amino Acid transport

4-iodo-L-meta-tyrosine,

L selectivesystem L. Chemistry & biology. 2006;13:1153-1160

artificial amino acid: molecular design and transport18) Giglia JL, White MJ, Hart AJ, Toro JJ, Freytes CO,

characterization in Chinese hamster ovary cells

Holt CC, Cai Y, Williams SM, Brandt SJ. A single(CHO-K1 cells). Nucl Med Biol. 2010; 37:903-910.

nucleotide polymorphism in SLC7A5 is associatedasystem

11) Shikano N, Kanai Y, Kawai K, Inatomi J, Kim DK,

withgastrointestinal

toxicity

after

high-dose

Ishikawa N, Endou H. Isoform selectivity ofmelphalan and autologous stem cell transplantation

3-125I-iodo-alpha-methyl-L-tyrosine

formembranetransport in human L-type amino acid transporters. J12) Yanagida O, Kanai Y, Chairoungdua A, et al. Humanaminoacidtransporter

myeloma.

BiolBloodMarrowTransplant. 2014;20:1014-1020.
19) Wongthai P, Hagiwara K, Miyoshi Y, Wiriyasermkul

Nucl Med. 2003; 44:244-246.
L-typemultiple

1P, Wei L, Ohgaki R, Kato I, Hamase K, Nagamori S,(LAT1):

Kanai Y. Boronophenylalanine, a boron delivery

characterization of function and expression in tumor

agent for boron neutron capture therapy, is transported

cell lines. Biochim Biophys Acta. 2001;1514:291-by302.

2015;106:279-286.

ATB0,+,

LAT1

andLAT2.

CancerSci.

13) Segawa H, Fukasawa Y, Miyamoto K, Takeda E,20) Wei L, Tominaga H, Ohgaki R, Wiriyasermkul P,Endou H, Kanai Y. Identification and functional

Hagiwara K, Okuda S, Kaira K, Oriuchi N, Nagamori

characterization of a Na-independent neutral amino

S, Kanai Y. Specific transport of 3-fluoro-l-α-methyl-acid transporter with broad substrate selectivity. Jtyrosine by LAT1 explains its specificity to malignant

Biol Chem. 1999;274:19745-19751.

tumors in imaging. Cancer Sci. 2016;107:347-352.

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茨城県立医療大学紀要28

第 24 巻

和文抄録
我々はこれまでに，L-type amino acid transporter-1 (hLAT1) に対する 3-125I- α -methyl-L-tyrosine (125I-IMT)の高いアイソフォーム選択性を報告した。今回，さらに他の hLAT1 選択的化合物を探索するために 4-125I-Lmeta-tyrosine (4-125I-mTyr), 6-125I-L-meta-tyrosine (6-125I-mTyr)，3-125I-L-tyrosine (3-125I-Tyr) を，hLAT1 またはhLAT2 発現 Xenopus laevis 卵母細胞を用いて調べた。その結果，選択性の高い順は 4-125I-mTyr > 125I-IMT >3-125I-Tyr > L-14C(U)-Tyr ≑ 6-125I-mTyr であった。hLAT は，4-125I-mTyr に対して最も高い輸送特異性を示した。

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